Cancer specific oligosaccharide sequences and use thereof

ABSTRACT

The invention concerns a method for diagnosing cancer in a biological sample by determining the presence of a LacdiNAc oligosaccharide sequence. The invention can be used for diagnostic agents, pharmaceutical compositions, cancer vaccines, and antigenic carbohydrate substances. The presence of cancer and malignancies is determined by contacting the biological sample with a reagent that binds to the oligosaccharide sequence. This same method can also be used in the treatment of cancer and malignancies.

FIELD OF THE INVENTION

The present invention relates to oligosaccharide sequences, which arespecifically expressed by certain cancer cells, tumours and othermalignant tissues. The present invention describes methods to detectcancer specific oligosaccharide sequences as well as methods for theproduction of reagents binding to said oligosaccharide sequences. Theinvention is also directed to the use of said oligosaccharide sequencesand reagents binding to them for the diagnostics of cancer andmalignancies. Furthermore, the invention is directed to the use of saidoligosaccharide sequences and reagents binding to them for the treatmentof cancer and malignancies.

BACKGROUND OF THE INVENTION

Various cancer cells or tissues express oligosaccharide sequences whichare different from the non-malignant glycosylation of the same cell ortissue type. Examples of the known or speculated cancer associatedoligosaccharide structures include: glycolipid structures such asglobo-H (Fucα2Galβ3GalNAcβ3Galα4LacβCer), ganliosides: GM1Galβ3GalNAcβ4(NeuNAcα3)LacβCer, or GD2GalNAcβ4(NeuNAcα8NeuNAcα3)LacβCer; Lewis-type fucosylated structuressuch as Lewis a and x Galβ3/4(Fucα4/3)GlcNAc, Lewis yFucα2Galβ4(Fucα3)GlcNAc, sialyl-Lewis x NeuNAcα3Galβ4(Fucα3)GlcNAc, andsome combinations of these on polylactosamine chains; O-glycan corestructures such as T-antigen Galβ3GalNAcαSer/Thr-Protein, Tn-antigenGalNAcαSer/Thr-Protein or sialyl Tn-antigenNeuNAcα6GalNAcαSer/Thr-Protein. Presence of non-human structures such asN-glycolyl-neuraminic acid in cancers has also been indicated.Association and specificity of oligosaccharide structures with regard tocancers have been well established only in few cases, some of thestructures are present in normal cells and tissues and are possibly onlymore concentrated in cancers. However, absolute cancer specificity isprobably not always needed for therapeutic applications.

LacdiNAc (GalNAcβ4GlcNAc)-type glycosylations have not been found to becommonly present on human tissues. However, LacdiNAc-type saccharidesequences have been reported from many non-human animals, bovineglycoproteins, human glycoprotein hormones (Manzella et al., 1997) andhuman glycodelin protein, reviewed in van den Eijnden et al. (1997).Generally the structure seem to be associated with invertebrate animalsand early development. Several LacdiNAc variants has been reported fromproteins expressed in human embryonal kidney 293 cells (Do et al.,1997). Recently the inventors described LacdiNAc-based structures fromtransfected fibroblast cells (Saarinen et al., 1999). This study did notshow if the glycosylation is related to cancer or to the transfection ofthe adenoviral EIA-promoter sequence to the cells as EIA-promoter mayregulate the gene expression of glycosyltransferases and thereby modifythe glycosylation. LacdiNAc type saccharides were also detected amongother structures from tissue type plasminogen activator of Bowesmelanoma cells, but these were considered to be “nervous systemassociated” structures (Jaques et al., 1996) The previous studies alsodescribe the detection of similar oligosaccharide structures from celllines derived from solid tumors (Do et al., 1997; Jaques et al., 1996;Saarinen et al., 1999). However, carbohydrate and other cell surfaceantigens usually change when contacts between cells are changed, forinstance, when a solid tumor is divided to single cells. Besides, cellsof cell lines are possibly genetically modified and only then culturedas single cells. Cell surface glycosylations are also very specific forthe differentiation status of a cell line or tissue and they arespecific for a cell or tissue type. Therefore prior art discussed hereindo not describe the natural glycosylation status of a single cancer cellor solid tumor tissue. However, the potential correlations of theglycosylations with cell type or differentiation status allow the use ofthe cancer antigen(s) for the typing of cancer cells and tumors.

The following patents describe cancer antigens and their use for makingantibodies for therapeutic and diagnostic uses and for cancer vaccines.The antigen structures are not related to saccharides of the presentinvention:

-   Cancer vaccines: U.S. Pat. No. 5,102,663 describes composition    comprising 9-OAc NeuNAcα8NeuNAcα3Lac-Cer (GD3) for the stimulation    or the enhancement of the production of antibodies against 9-OAc    GD3.-   U.S. Pat. No. 5,660,834 describes pharmaceutical composition    containing mucin type glycoprotein consisting essentially of Tn    (GalNAcα-Ser/Thr) or sialyl-Tn antigens (NeuNAcα6GalNAcα-Ser/Thr)    and uses thereof with adjuvant to reduce cancer cell growth rate.    Inventions related to the same mucin sequences are also described in    other patents: U.S. Pat. No. 5,747,048 (adjuvant therapy for human)    and U.S. Pat. No. 5,229,289.-   U.S. Pat. No. 6,083,929 describes extended type 1 chain    sphingolipids (Galβ3GlcNAc) as tumour-associated compositions and    pharmaceutical composition with an adjuvant.

Therapeutic antibodies: U.S. Pat. No. 4,851,511 describes a monoclonalantibody that bind disialosyl Lewis a-structureNeuNAcα3Galβ3(Fucα4)[NeuNAcα6]GlcNAc, diagnostic test kits, hybridomasproducings antibodies, marker molecules and an antitumor drug conjugatedwith antibodies.

U.S. Pat. No. 4,904,596 describes a monoclonal antibody that bindsstructure NeuNAcα3Galβ4(Fucα3)GlcNAcβ3Galβ4(Fucα3)GlcNAcβ3LacCer,hybridomas, diagnostics, and coupling of the antibody to an antitumordrug, an immunoregulatory agent or a differentiation inducing agent.

U.S. Pat. No. 5,874,060 describes humanized antibodies recognizing Lewisy-antigen Fucα2Galβ4(Fucα3)GlcNAc.

U.S. Pat. No. 6,025,481 describes nucleic acid molecules encodinghumanized antibodies recognizing Lewis b-antigen. The Lewis b-structureFucα2Galβ3(Fucα4)GlcNAc-expression is increased in cancer cells. U.S.Pat. No. 5,795,961 describes also anti-Lewis b antibodies.

Diagnostics: U.S. Pat. No. 4,725,557 describes protein linked antigensFucα3Gal-, Fucα4Gal- and Fucα6Gal-, and antibodies recognizing thesestructures, method of determining the cancer associated carbohydratelinkages and diagnostic kits. The antibodies bind cancer cells of humandigestive system.

U.S. Pat. No. 5,059,520 describes several monoclonal antibodiesrecognizing blood group A-antigen GalNAcα3(Fucα2)Galβ-, which may beused for cancer diagnostics.

U.S. Pat. No. 5,171,667 describes antibodies against fucosylated type 2lactosamines (-Galβ4(Fucα3)GlcNAcβ-) and use thereof for cancerdiagnostics.

U.S. Pat. No. 5,173,292 describes monoclonal antibodies binding toGal-globoside, Galβ3GalNAcβ3Galα4LacCer, which is a cancer specificstructure.

U.S. Pat. Nos. 6,090,789 and 5,708,163 describe synthesis ofFucα2Galβ3GalNAcβ3Galα4LacCer (Globo H, MBr1, breast tumor associatedantigen) conjugates and analogs thereof, and pharmaceutical compositionscontaining the same. U.S. Pat. No. 5,679,769 describes the synthesis ofasparagines linked to glycopeptides. U.S. Pat. No. 5,543,505 describessynthetic compounds which bind Helicobacter pylori bacteria.

U.S. Pat. Nos. 5,902,725 and 6,203,999 describe the detection ofprostate specific cancer by assaying at least triantennaryoligosaccharides on prostate specific antigen. Antibody or lectin PHA-Lis used for detection. The patents characterize chromatographically thepresence of triantennary N-glycans on cancer form of PSA from a cellline.

The prior art describes fucosylated lacdiNAcs containing relativelylarge N-glycan structures (EP0565241) and a core 2-type O-glycanstructures (EP0919563) in pharmaceutical compositions. The compositionsare aimed for inhibition of selectin mediated cell adhesion and inEP0919563 also for inhibition of metastasis by inhibiting selectinmediated cell adhesion. However, the present invention is directed tothe use of the oligosaccharide epitopes according to the presentinvention as targets of specific recognition molecules, includingantibodies. The present invention is specifically directed to suitableantigenic conjugates and compositions for inducing antibodies fordiagnostics and therapies. The present invention is directed topharmaceutical compositions comprising optimal size of oligosaccharidesequences for recognition by specific antibodies. The optimaloligosaccharide epitopes may comprise only the terminal lacdinacstructure or the terminal oligosaccharide sequence and one or twomonosaccharide residues.

SUMMARY OF THE INVENTION

The present invention describes oligosaccharide sequences, which arespecifically expressed by cancer cells. The present invention is relatedto a method of determining an oligosaccharide sequence, which comprisesa cancer specific sequence of Formula(Sac1)_(x)GalNAcβ4(Fucα3)_(y)GlcNAc  (I),wherein x and y are each independently 0 or 1 and Sac1 is NeuNAcα3 orNeuNAcα6, in a biological sample, the presence of said sequence in saidsample being an indication of the presence and/or type of cancer. Thepresent invention provides antigenic substances comprising saidoligosaccharide sequences in a polyvalent form and it further providesdiagnostic agents, pharmaceutical compositions and cancer vaccinescomprising said oligosaccharide sequences or substances binding to saidoligosaccharide sequences. The present invention is also related tomethods for the treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C and 1D. MALDI TOF MS analysis of liberated MMP-9N-glycans. (1A) intact N-glycans; (1B) the N-glycans after incubationwith NDV neuramimidase, followed with consecutive treatments with (1C)C. perfringens neuraminidase and (1D) Almond meal fucosidase.

FIG. 2. LC-ESI MS analysis of tryptic digest of MMP-9. Panel A) showstotal ion chromatogram of eluting peptides; Panel B) shows extracted ionchromatogram of m/z 0.204.1 (Oxonium ion of Hex); Panel C) showsextracted ion chromatogram of m/z 292.1 (Oxonium ion of SA); and PanelD) shows extracted ion chromatogram of m/z 366.1 (Oxonium ion ofHex-HexNac). Panels B, C and D show putative glycopeptides.

FIGS. 3A and 3B. LC/MS analysis of tryptic peptides from MMP-9. (3A)Mass spectrum corresponding to glycopeptides eluting at 23.1 minutes,(3B) mass spectrum corresponding to glycopeptides eluting at 24.3minutes.

FIG. 4A. Negative ion linear mode MALDI-TOF mass spectrum of larynxcancer sample sialylated glycans.

FIG. 4B. Positive ion reflector mode MALDI-TOF mass spectrum of larynxcancer sample sialylated glycans after A. ureafaciens sialidase and S.pneumoniae β-N-acetylglucosaminidase digestions.

FIG. 4C. Positive ion reflector mode MALDI-TOF mass spectrum of larynxcancer sample sialylated glycans after A. ureafaciens sialidase, S.pneumoniae β-N-acetylglucosaminidase, and Jack beanβ-N-acetylhexosaminidase digestions.

FIG. 5. Positive ion reflector mode MALDI-TOF mass spectrum of RPMI-7932melanoma cell line membrane protein sialylated N-glycans after A.ureafaciens sialidase and S. pneumoniae β-N-acetylglucosaminidasedigestions.

FIG. 6. Positive ion reflector mode MALDI-TOF mass spectrum of RPMI-7932melanoma cell line membrane protein sialylated N-glycans after A.ureafaciens sialidase, S. pneumoniae β-N-acetylglucosaminidase, and Jackbean β-N-acetylhexosaminidase digestions.

FIG. 7. Positive ion reflector mode MALDI-TOF mass spectrum of RPMI-7932melanoma cell line membrane protein sialylated N-glycans after A.ureafaciens sialidase, Jack bean β-N-acetylhexosaminidase, andXanthomonas sp. α1,3/4-fucosidase digestions.

FIG. 8. Positive ion reflector mode MALDI-TOF mass spectrum of RPMI-7951melanoma cell line membrane protein sialylated N-glycans after A.ureafaciens sialidase and S. pneumoniae β-N-acetylglucosaminidasedigestions.

FIG. 9. Positive ion reflector mode MALDI-TOF mass spectrum of RPMI-7951melanoma cell line membrane protein sialylated N-glycans after A.ureafaciens sialidase, S. pneumoniae β-N-acetylglucosaminidase, and Jackbean β-N-acetylhexosaminidase digestions.

DETAILED DESCRIPTION OF THE INVENTION

Several lacdiNAc (GalNAcβ4GlcNAc) type sequences are known fromsecretory proteins such as glycoprotein hormones. In human glycoproteinhormones the lacdiNAc is usually modified to4sulfo-GalNAcβ4GlcNAc-sequences which are considered to be important forthe hormone function, however, some heterogeneity exist in the secretedlacdiNAc-structures. The glycoprotein hormones are rare solubleproteins, relatively small in general and contain few glycosylationsites with quite limited selection of lacdiNAc structures. The lacdiNAcsequence has not been structurally characterized on humanmembrane-linked non-secreted proteins, though numerous structuralstudies of human glycosylations have carried out during last 30 years.

The role of lacdiNAc structures as an intracellular secretion marker hasbeen demonstrated, the same report specifically states that lacdiNAcstructures are not present on membrane proteins of MDCK cells (Ohkura,et al. 2001). The lacdiNAc structures characterized from secretedproteins have not been shown to be related to human cancer. The unusualexpression of the lacdiNAc structures and especially rare fucosylatedand sialylated variants thereof by cancers, but not on normal tissues,offer unusually good possibilities for cancer treatment and therapiesbased on the recognition of the structures. Diagnostic and therapeuticsuccess have obtained by carbohydrates which are present on limitedamount of normal tissues or in low density on normal tissues and/or inserum.

Methods Combining Diagnostics and Therapy for Effective Cancer Treatment

The data of present invention shows that lacdiNAc structures accordingto the present invention are useful for use of diagnostic andtherapeutic methods which recognize the carbohydrate structures oncancer cells. As the glycosylation patterns varies between tissues,between different types of tumors and even between individual patients,the present invention is specifically directed to methods to screenunusual carbohydrate structures from tumors and direct individualtherapy against the forms of glycosylation specifically expressed on thecancer of a specific patient. The present invention is specificallydirected to the screening of lacdiNAc type glycosylations according tothe present invention on tumor or cancer samples and using lacdiNActargeting therapies according to the present invention for treatment ofa patient who has the specific cancer associated glycosylationspecifically on tumor or on malignant tissue or cells.

The inventors screened multiple normal tissues by effective massspectrometric methods to verify that lacdiNAc is not cell surface markeron normal tissues. In the present invention it was found that

-   (i) the lacdiNAc sequence occur in cancers both on unicellular    cancer such as leukaemia and on solid tumors,-   (ii) the lacdiNAc sequence was not observable on multiple on normal    tissues,-   (iii) the lacdiNAc sequence is present both on plasma membrane or    membrane associated proteins and    on secreted proteins of cancers, the integrated cell surface    lacdiNAc sequences are targets for cancer diagnostics and    immunotherapies and other therapies based to the recognition of the    lacdiNAc sequences on cancers, and-   (iv) the lacdiNAc sequences on cancer cell surfaces can be    recognized by specific antibodies for therapy and diagnostics. The    structures are available on cell surfaces and not covered by other    cell surface components.

Leukeamia cells were chosen as a model for unicellular cancer cells. Theleukeamia cells represent the unicellular cancer cells in the bloodcancers. The soluble target protein was chosen so that structuralcomparison data from non-malignant cells exists. To demonstrate abnormalexpression of LacdiNAc sequence on cancer cells metalloproteinase-9(MMP-9) was isolated from leukaemia cells (U-937), and N-glycosidicglycans were liberated with N-glycosidase F. The glycan fraction wasanalyzed with MALDI-TOF MS in trihyxroacetophenone matrix (FIG. 1A),which has been shown to cause negligible fragmentation of sialic acidresidues. The assignment of the monosaccharide compositions as well asthe proposed structures according to the subsequent glycosidasetreatments are shown in Table 1. The relative abundances of thecomponents are indicated as well, as oligosaccharide analysis inMALDI-TOF has been shown to be relatively quantitative. The mostabundant glycan species was assigned to [M+Na]⁺ of(Hex)₅(HexNAc)₄(Fuc)₃. Knowing the typical N-glycan structures, wetentatively assigned this structure as a trifucosylated diantennarycomplex-type glycan. Other major species were identified as sialylated,difucosylated diantennary complex-type glycan, and trifucosylateddiantennary complex-type glycan carrying GalNAc instead of Gal as aterminal monosaccharide (so called LacdiNAc structure) in one antennae.These assignments were found to be correct, as shown by sequentialglycosidase treatments. For comparison the matrix metalloproteinaseMMP-9 structures in non-malignant white blood cells (leukocytes) havebeen determined, the protein did not contain lacdiNAc sequences (Rudd Pet al, 1999).

The present invention shows that U-937-cell derived MMP-9 carriesLacdiNAc structures in large fraction (approximately 30%) of itsN-glycans. The presence of LacdiNAc structures was verified by twoindependent methods, namely MALDI-TOF analysis of liberated N-glycans aswell as LC-ESI MS of intact glycopeptides. The assignments of thestructures were further confirmed by sequential glycosidase treatments.The methods used in this study have been verified by several approachesusing both known natural structures as well as syntheticoligosaccharides.

Mass Spectrometric Screening of lacdinac-Structures from Normal Tissues

Membrane glycoprotein samples of several non-malignant tissues,including human stomach, lungs, and colon were analyzed massspectrometric methods as described above. No N-linked or O-linked typelacdiNAc-sequences were observed. The prior art does not describelacdiNac on human normal or cancer membrane proteins either.

Analysis of lacdiNAc Structures on Solid Tumors

The present invention demonstrates also for the first time thatlacdiNAc-sequences are present on human solid tumors. An example of thepresent inventions shows characterization of lacdiNAc structure fromsample of cancer of human larynx.

Characterization of lacdiNAc Sequences from Plasmamembrane Samples

The present invention is also directed to the plasmamembrane forms oflacdiNAc expressing proteins on cancers. The matrix metalloproteinaseMMP-9 is also known to occur as membrane associated form (Koivunen etal., 1999), which according to present invention also form ideal targetfor cancer diagnostics and immune therapy. As another example,glycosylations of two different samples of melanoma related membraneswere analyzed. Large amounts of various lacdiNAc-type oligosaccharidesequences were found on the membrane bound glycoproteins includingunique N-glycan structures. The glycosylated membrane proteins are knownto be presented on the surface of the cells and tissues.

Specific Defect in Cancer Demonstrated by the Present Invention

The present invention shows a novel defect in cancer cells. The rarestructure associated with secretory proteins is expressed on proteinswhich normally do not express the lacdiNAc structures. Furthermore thefailure in glycosylation induces more unusual sialylated, fucosylated,and variants without usual sulphation on position 4 of GalNAc. A generalunderstanding about the cancers are that the intracellular organizationof cancer cells is disturbed. Such errors in Golgi apparatus whichproduces very specific glycosylations on different cell types obviouslyleads to the problems described. Obviously presence of the cancerindicating abnormal glycosylations on soluble proteins is useful forcancer diagnostics, the presence of the cancer glycosylation associatedwith membranes makes these directly useful for therapeutics.

As described above the presence of cancer antigens was studied fromsecreted MMP-9 protein of leukaemia cell line U-937 from solid tumors,and from membrane preparations. The single cells of a leukemia cell lineare also considered to be a reasonably relevant model of single cellcancer leukaemia. Demonstration of the lacdiNAc-structures from solidtumors and being membrane associated shows the usefulness of the cancerglycosylation for therapies against solid tumors expressing theglycosylation.

The present invention shows that LacdiNAc structures according toFormula(Sac1)_(x)GalNAcβ4(Fucα3)_(y)GlcNAc  (I)wherein x and y are each independently 0 or 1 and Sac1 is NeuNAcα3 orNeuNAcα6, are cancer specific antigens. The invention is directed to thedetection of the cancer antigens directly from cancer cells and tumortissues as a cell-associated form of MMP-9 is known (Koivunen et al.,1999). However, the cancer antigens may, in addition to the detectionfrom cancer cells and tumor tissues, be detected, as described herein,on a secreted glycoprotein derived from cancer cells or tissues. Thecancer specific proteins can be proteinases, hormones or secreted mucintype glycoproteins.

The present invention shows that a cancer antigen can be detected from aglycoprotein, which is known to be upregulated upon malignanttransformation. The presence of the cancer antigen on a putative cancerassociated glycoprotein may provide a more reliable diagnostic tool ofcancer in early phase. Examples of such preferred cancer associatedproteins include, but are not limited to, members of matrixmetalloproteinase protein family (e.g. MMP-9), prostate specificantigen, kallikrein 2, human chorionic gonadotrophin andcarcinoembryonic antigen.

Cancer specific oligosaccharides of the LacdiNAc type containsGalNAcβ4GlcNAc-oligosaccharide sequence. The sequence is a part of anoligosaccharide glycoconjugate of cancer cells. The oligosaccharidesequence can be substituted to sequence GalNAcβ4(Fucα3)GlcNAc-,NeuNAcα3GalNAcβ4GlcNAc-, NeuNAcα6GalNAcβ4GlcNAc-,NeuNAcα3GalNAcβ4(Fucα3)GlcNAc-, or NeuNAcα6GalNAcβ4(Fucα3)GlcNAc-, whichwere also indicated to be present on the cancer cells. If a singlecancer oligosaccharide epitope contains both sialic acid and fucose thestructure can be NeuNAcα3GalNAcβ4(Fucα3)GlcNAc-. The LacdiNAc sequencecan also be sulphated, for instance, to position 4 of GalNAc, if thecancer cells contain a sulphotransferase needed for the modification.The LacdiNAc-type sequence(s) can be a part of a glycoprotein sequenceof cancer cells or tissue, for instance, LacdiNAc type sequence is β2-,β4- or β6-linked to a mannose residue in a N-linked glycan of aglycoprotein, or the LacdiNAc-sequence is β2-linked to a mannose residuein a N-linked glycan of a glycoprotein.

The fucosylated LacdiNAc saccharides are analogous to Lewis type cancerassociated oligosaccharides. Potential weak cross reactivity withfucosylated LacdiNAcs is a probable explanation for the production ofhuman antibodies weakly recognizing Lewis type cancer associatedoligosaccharides though these are present in large amounts also innormal tissues.

The present invention also describes methods to detect malignancy of acell or a tissue by detecting cancer specific glycosylations. Thedetection can be performed by molecules specifically binding to thecancer specific oligosaccharide sequences of the invention. Preferablythe molecules specifically binding to the cancer specificoligosaccharide sequences are aptamers, lectins, genetically engineeredlectins, antibodies, monoclonal antibodies, antibody fragments, enzymesrecognizing LacdiNAc-structure such as glycosidases andglycosyltransferase and genetically engineered variants thereof.Labelled bacteria, viruses or cells or other polymeric surfacescontaining molecules recognizing the structures can be used for thedetection. Oligosaccharide sequences can also be released from cancercells by endoglycosidase enzymes. Alternatively oligosaccharides can bereleased as glycopeptides by protease enzymes. Chemical methods torelease oligosaccharides or derivatives thereof include, e.g.,otsonolysis of glycolipids and beta-elimination or hydrazinolysismethods to release oligosaccharides from glycoproteins. Alternativelythe glycolipid fraction can be isolated. A substance specificallybinding to the cancer specific oligosaccharide sequences can also beused for the analysis of the same sequences on cell surfaces. Saidsequences can be detected, e.g., as glycoconjugates or as releasedand/or isolated oligosaccharide fractions. The possible methods for theanalysis of said sequences in various forms also includeNMR-spectroscopy, mass spectrometry and glycosidase degradation methods.Preferably at least two analysis methods are used, especially whenmethods of limited specificity are used.

Mass spectrometry is a preferred method to determine the cancer specificoligosaccharide sequence or sequences according to the invention in asample. Mass spectrometric scanning methods for the detection ofHexNAc-HexNAc-fragments from a fraction containing oligosaccharidesequences according to Formula I are especially preferred.

The present invention is also directed to the use of cancer specificoligosaccharide sequences or analogs or derivatives thereof to producepolyclonal or monoclonal antibodies recognizing the structures usingfollowing process: 1) producing synthetically or biosynthetically apolyvalent conjugate of an oligosaccharide sequence of the invention oran analogue or derivative thereof, the polyvalent conjugate being, forinstance, according to the following structure: position C1 of reducingend terminal of the oligosaccharide sequence (OS) comprising the cancerspecific terminal sequence of the invention is linked (-L-) to anoligovalent or a polyvalent carrier (Z), via a spacer group (Y) andoptionally via a monosaccharide or oligosaccharide residue (X), forminga structure according to Formula[OS—(X)_(n)-L-Y]_(m)—Z  (II),where integer m has values m>1, and n is independently 0 or 1; L isoxygen, nitrogen, sulfur or carbon atom, X is preferably lactosyl-,galactosyl-, poly-N-acetyl-lactosaminyl, or part of an O-glycan or anN-glycan oligosaccharide sequence, Y is a spacer group or a terminalconjugate such as a ceramide lipid moiety or a linkage to Z; preferablyone of the following properties are present: the oligosaccharidesequence (OS) is sialylated, X comprises at least one mannose orN-acetylgalactosamine residue or Z comprises a carbohydrate material,such as a polysaccharide; 2) immunizing an animal or human with thepolyvalent conjugate together with an immune response activatingsubstance. Preferably the oligosaccharide sequence is polyvalentlyconjugated to the immune response activating substance and the conjugateis used for immunization alone or together with an additional immuneresponse activating substance. In a preferred embodiment theoligosaccharide conjugate is injected or administered mucosally to anantibody producing organism with an adjuvant molecule or adjuvantmolecules. For antibody production the oligosaccharide or analogs orderivatives thereof can be polyvalently conjugated to a protein such asBSA, keyhole limpet hemocyanin, a lipopeptide, a peptide, a bacterialtoxin, a part of peptidoglycan or immunoactive polysaccharide or toanother antibody production activating molecule. The polyvalentconjugates can be injected to an animal with adjuvant molecules toinduce antibodies by routine antibody production methods known in theart. Preferably an antigenic substance of the invention comprises aterminal oligosaccharide sequence as defined in Formula I in achemically or biochemically synthezised polyvalent form described abovefor immunization in human. More preferably the antigenic substancecomprises terminal NeuNAcα3 or NeuNAcα6 (i.e. x=1 in Formula I) or thesaccharide sequence is linked to mannose or N-acetylgalactosamine (e.g.X is Man or GalNAc in Formula II).

The present invention is also directed to monovalent and/oligovalentantigenic conjugates of oligosaccharide sequences according to thepresent invention. Monovalent antigenic conjugate may comprise anantigenic lipid structure, for example a ceramide, a synthetic lipid ora bacterial type of lipid which can induce antibody production asdescribed by the invention and by methods known in the art.

The present invention is specifically directed to the use of optimalsize antigenic epitopes and pharmaceutical compositions comprisingthese. Antibodies can usually recognize effectively only epitopes of afew monosaccharide residues. Reduced size of the epitope is alsopreferred because of more cost effective synthesis of the structures.

Preferred optimal antigenic epitopes includes structures according tothe Formula II[OS—(X)_(n)-L-Y]_(m)—Z  (II),wherein Y is a non-carbohydrate spacer or a non-glycosidically linkedterminal conjugate, n is 0 or 1 and X is lactosyl-, galactosyl-,N-acetyllactosaminyl, mannosyl-, Man₂, Man₃-, Man₃GlcNAc, Man₄GlcNAc,N-acetylglucosaminyl-, or N-acetylgalactosaminyl. More preferably X islactosyl-, galactosyl-, mannosyl-, or N-acetylgalactosaminyl. In apreferred embodiment the OS is β2-, or β4, or β6 linked to themannosylresidue, most preferably β2-. In a preferred embodiment the OSis β3- or β6-linked to galactosylresidue orN-acetylgalactosaminylresidue or Gal-residue of lactose orN-acetylalactosamine, more preferably β3- or β6-linked to Gal or Gal oflactose or to GalNAc, and most preferrably β3-linked to Gal or Gal oflactose or β6-linked to GalNAc. In a preferred embodiment the optimalantigenic epitopes as described above does not comprise fucose in theoligosaccharide sequence.

Man₂, Man₃-, Man₃GlcNAc, Man₄GlcNAc indicates preferably parts ofN-glycan core structures comprising oligosaccharide sequences Manα3Man,Manα6Man, Manα3(Manα6)Man, Manα3(Manα6)Man, Manα3(Manα6)Manα4GlcNAc andhydrid type of structure wherein additional mannose is linked to eithernon-reducing terminal Man, preferably Manα6 branch, and theoligosaccharide sequence according to the present invention to the otherbranch of the molecule.

In a preferred embodiment optimal antigenic epitope comprisingstructures OSβ2Man, OSβ2Manα3Man, or OSβ2Manα6Man are used. These arepartial epitopes of the N-glycan lacdiNAc primarily observed by thepresent invention. In a separate embodiment it is also realized thatbecause of the glycosylation defects in cancer cells, O-glycan typelacdiNAc and partial lactosamine type lacdiNAcs comprising similaroptimal antigenic epitopes OSβ3Gal, OSβ3GalNAc and especially OSβ6Gal,OSβ6GalNAc are useful for immunization and other uses according to thepresent invention against tumors comprising these structures. Suchtumors are characterized by lacdiNAc type secretory functions combinedwith lactosamine and or mucin production.

The cancer specific oligosaccharides or derivatives or analogs thereofcan be immobilized for the purification of antibodies from serum,preferably from human serum. The cancer specific oligosaccharides,preferably as polyvalent conjugates, can also be used for the detectionand/or quantitation of antibodies binding to these cancer specificoligosaccharides, for example, in enzyme-linked immunosorbent assay(ELISA) or affinity chromatography type assay formats for thediagnostics of cancer.

Antibody production or vaccination can be also achieved by analogs orderivatives of the cancer specific oligosaccharide sequences. Simpleanalogs of the N-acetyl-group containing oligosaccharide sequencesinclude compounds with modified N-acetyl groups, for example, N-acylsuch as N-propanoyl. The present invention is also directed forproduction of specific analogs for the cancer specific oligosaccharidesequences as described by the invention.

Furthermore, it is possible to use human antibodies or humanizedantibodies against the cancer specific oligosaccharide sequences toreduce the growth of or to destroy a tumor or cancer. Human antibodiescan also be tolerated analogs of natural human antibodies against thecancer specific oligosaccharide sequences; the analogs can be producedby recombinant gene technologies and/or by biotechnology and they may befragments or optimized derivatives of human antibodies. Purified naturalanti-tumor antibodies can be administered to a human without anyexpected side effect as such antibodies are transferred during regularblood transfusions. This is true under conditions that the cancerspecific structures are not present on normal tissues or cells and donot vary between individuals as blood group antigens do, however, suchblood-group-like variations are not known for the cancer specificoligosaccharide sequences of the invention. In another embodiment of theinvention species specific animal antibodies are used against a tumor orcancer of the specific animal. The production of specific humanizedantibodies by gene engineering and biotechnology is also possible: theproduction of humanized antibodies has been described in U.S. Pat. Nos.5,874,060 and 6,025,481, for example. The humanized antibodies aredesigned to mimic the sequences of human antibodies and therefore theyare not rejected by immune system as animal antibodies are, ifadministered to a human patient. It is realized that the method toreduce the growth of or to destroy cancer applies both to solid tumorsand to cancer cells in general. It is also realized that the purifiednatural human antibodies recognizing any human cancer specific antigen,preferably an oligosaccharide antigen, can be used to reduce the growthof or to destroy a tumor or cancer. In another embodiment speciesspecific animal antibodies are used against a tumor or cancer of thespecific animal.

According to the invention human antibodies or humanized antibodiesagainst the cancer specific oligosaccharides, or other toleratedsubstances binding the cancer specific oligosaccharides, are useful totarget toxic agents to tumor or to cancer cells. The toxic agent couldbe, for example, a cell killing chemotherapeutics medicine, such asdoxorubicin (Arap et al., 1998), a toxin protein, or a radiochemistryreagent useful for tumor destruction. Such therapies have beendemonstrated and patented in the art. The toxic agent may also causeapoptosis or regulate differentiation or potentiate defence reactionsagainst the cancer cells or tumor. In another embodiment of theinvention species specific animal antibodies are used against a tumor orcancer of the specific animal. The cancer binding antibodies accordingto the present invention can also be used for targeting prodrugs activeagainst cancer or enzymes or other substances converting prodrugs toactive toxic agents which can destroy or inhibit cancer, for example inso called ADEPT-approaches.

The therapeutic antibodies described above can be used in pharmaceuticalcompositions for the treatment or prevention of cancer. The method oftreatment of the invention can also be used when patient is underimmunosuppressive medication or he/she is suffering fromimmunodeficiency.

Immunosuppressive medications are used, for instance, with organtransplantations to prevent rejection during kidney, heart, liver orlung transplantations. Malignancies arising during such therapies are ingeneral benign, but they cause often the loss of the precious organtransplant. Capability of producing antibodies against the tumor orcancer specific antigens may vary according to individual differences inimmune system. Persons who have survived from cancer may have especiallyhigh amounts of natural anti-cancer antibodies.

Other Methods for Therapeutic Targeting of Cancers

It is realized that numerous other agents beside antibodies, antibodyfragments, humanized antibodies and the like can be used fortherapheutics targeting cancers similarly with the diagnosticsubstances. It is specifically preferred to use non-immunogenic andtolerable substances to target cancer. The targeting substances bindingto the cancer comprise also specific toxic or cytolytic or cellregulating agents which lead to destruction or inhibition of cancer.Preferably the non-antibody molecules used for cancer targetingtherapies comprise molecules specifically binding to the cancer specificoligosaccharide sequences according to the present invention areaptamers, lectins, genetically engineered lectins, enzymes recognizingLacdiNAc-structure such as glycosidases and glycosyltransferase andgenetically engineered variants thereof. Labelled bacteria, viruses orcells or other polymeric surfaces containing molecules recognizing thestructures can be used for the cancer targeting therapies. The cancerbinding non-antibody substances according to the present invention canalso be used for targeting prodrugs active against cancer to cancers orenzymes or other substances converting prodrugs to active toxic agentswhich can destroy or inhibit cancer.

The present invention is specifically directed to the use of substancesand antibodies binding to cancer specific oligosaccharide sequencesaccording to the present invention for therapies in gastrointestinaltract of the patient, preferably in human patient. The therapeuticantibodies for use in human gastrointestinal tract may be antibodiesproduced by animals for example antibodies in milks of domestic animals,for example in milk of domestic ruminants such as cows, sheep, goat orbuffalo or antibodies produced in hen eggs. The animals can be immunizedby cancer specific carbohydrate conjugates as known in the art. Thepresent invention is also directed to other acceptable, preferably foodacceptable proteins which can be used in inhibition or destruction ofcancers in human gastrointestinal tract, such substances include plantlectins which are specific for the cancer specific oligosaccharidesequences. The present invention is directed to functional foods andfood additives containing antibodies recognizing the cancer specificoligosaccharide sequences according to the present invention ingastrointestinal tract, the present invention is also directed to theuse of other food acceptable substances especially lectins binding tothe cancer specific oligosaccharide sequences of gastrointestinal tractin functional foods or as food additives.

Furthermore according to the invention the cancer specificoligosaccharide sequences or analogs or derivatives thereof can be usedas cancer vaccines in human to stimulate immune response to inhibit oreliminate cancer cells. The treatment may not necessarily cure cancerbut it can reduce tumor burden or stabilize a cancer condition and lowerthe metastatic potential of cancers. For the use as vaccines theoligosaccharides or analogs or derivatives thereof can be conjugated,for example, to proteins such as BSA or keyhole limpet hemocyanin,lipids or lipopeptides, bacterial toxins such as cholera toxin or heatlabile toxin, peptidoglycans, immunoreactive polysaccharides, or toother molecules, cells or cell preparations activating immune reactionsagainst a vaccine molecule. A cancer vaccine may also comprise apharmaceutically acceptable carrier and optionally an adjuvant. Suitablecarriers or adjuvants are, e.g., lipids known to stimulate the immuneresponse. The saccharides or derivatives or analogs thereof, preferablyconjugates of the saccharides, can be injected or administeredmucosally, such as orally or nasally, to a cancer patient with toleratedadjuvant molecule or adjuvant molecules. The cancer vaccine can be usedas a medicine in a method of treatment against cancer. Preferably themethod is used for the treatment of a human patient. Preferably themethod of treatment is used for the treatment of cancer of a patient,who is under immunosuppressive medication or the patient is sufferingfrom immunodeficiency.

Furthermore it is possible to produce a pharmaceutical compositioncomprising the cancer specific oligosaccharide sequences or analogs orderivatives thereof for the treatment of cancer. Preferably thepharmaceutical composition is used for the treatment of a human patient.Preferably the pharmaceutical composition is used for the treatment ofcancer, when patient is under immunosuppressive medication or he/she issuffering from immunodeficiency. The methods of treatment or thepharmaceutical compositions described above are especially preferred forthe treatment of cancer diagnosed to express the cancer specificoligosaccharide sequences of the invention. The methods of treatment orthe pharmaceutical compositions can be used together with other methodsof treatment or pharmaceutical compositions for the treatment of cancer.Preferably the other methods or pharmaceutical compositions comprisecytostatics, anti-angiogenic pharmaceuticals, anti-cancer proteins, suchas interferons or interleukins, or a use of radioactivity.

Use of antibodies for the diagnostics of cancer and for the targettingof drugs to cancer has been described with other antigens andoligosaccharide structures (U.S. Pat. No. 4,851,511; U.S. Pat. No.4,904,596; U.S. Pat. No. 5,874,060; U.S. Pat. No. 6,025,481; U.S. Pat.No. 5,795,961; U.S. Pat. No. 4,725,557; U.S. Pat. No. 5,059,520; U.S.Pat. No. 5,171,667; U.S. Pat. No. 5,173,292; U.S. Pat. No. 6,090,789;U.S. Pat. No. 5,708,163; U.S. Pat. No. 5,902,725 and U.S. Pat. No.6,203,999). Use of cancer specific oligosaccharides as cancer vaccineshas also been demonstrated with other oligosaccharide sequences (U.S.Pat. No. 5,102,663; U.S. Pat. No. 5,660,834; U.S. Pat. No. 5,747,048;U.S. Pat. No. 5,229,289 and U.S. Pat. No. 6,083,929).

The substance according to the invention can be attached to a carrier.Methods for the linking of oligosaccharide sequences to a monovalent ormultivalent carrier are known in the art. Preferably the conjugation isperformed by lining the cancer specific oligosaccharide sequences oranalogs or derivatives thereof from the reducing end to a carriermolecule. When using a carrier molecule, a number of molecules of asubstance according to the invention can be attached to one carrierincreasing the stimulation of immune response and the efficiency of theantibody binding. To achieve an optimal antibody production, conjugateslarger than 10 kDa carrying typically more than 10 oligosaccharidesequences are preferably used.

The oligosaccharide sequences according to the invention can besynthesized, for example, enzymatically by glycosyltransferases, or bytransglycosylation catalyzed by a glycosidase enzyme or atransglycosidase enzyme, for review see Ernst et al., 2000.Specificities of the enzymes and their use of co-factors such asnucleotide sugar donors, can be engineered. Specific modified enzymescan be used to obtain more effective synthesis, for example,glycosynthase is modified to achieve transglycosylation but notglycosidase reactions. Organic synthesis of the saccharides andconjugates of the invention or compounds similar to these are known(Ernst et al., 2000). Carbohydrate materials can be isolated fromnatural sources and be modified chemically or enzymatically intocompounds according to the invention. Natural oligosaccharides can beisolated from milks of various ruminants and other animals. Transgenicorganisms, such as cows or microbes, expressing glycosylating enzymescan be used for the production of saccharides.

It is possible to incorporate an oligosaccharide sequence according tothe invention, optionally with a carrier, in a pharmaceuticalcomposition, which is suitable for the treatment of cancer in a patient.Examples of conditions treatable according to the invention are cancersin which the tumor expresses one or more of the cancer specificoligosaccharides described in the invention. The treatable cancer casescan be discovered by detecting the presence of the cancer specificoligosaccharide sequences in a biological sample taken from a patient.Said sample can be, e.g., a biopsy or a blood sample.

It is possible to inhibit the formation of cancer antigens described inthe invention by specific inhibitors of LacdiNAc biosynthesis. Suchinhibitors maybe analogs of donor nucleotide UDP-GalNAc. Methods toproduce inhibitory analogs for glycosyltransferases have been describedin the art. Preferably the inhibitor has specificity towards LacdiNAcsynthezising GalNAc-transferase.

Alternatively an essentially non-antigenic chemically and/orenzymatically synthesised oligo- or polyvalent conjugate of the cancerspecific oligosaccharide sequences of the invention can be used toprevent the adhesion and/or growth of cancer cells. As the antigenicityof the conjugates would cause the removal of inhibitory oligomers orpolymers from blood circulation by antibodies. Examples of antigenicsubstances of the invention have been described above. A non-antigenicpolysaccharide conjugate can be constructed according to Formula II withthe proviso that X, Y or Z are not immunogenic. Preferably the molecularweight of the conjugate is under 50 kilodaltons (kDa) and morepreferably under 10 kDa. The oligosaccharide sequences of the inventioncan also be conjugated to a non-protein carrier. Preferably the cancerspecific oligosaccharide sequence is conjugated to a non-immunogenicpolysaccharide and most preferably the molecular weight of the conjugateis under 10 kDa.

As selectin carbohydrate interactions mediate cancer metastasis, theLacdiNAc-type glycosylations may target metastasing cancer cells toselectin containing sites on blood vessel endothelium as such structuresare known to be potent selectin ligands (Grinnel et al., 1994; Jain etal., 1998). LacdiNAc saccharides have immunomodulatory activities whichmay protect them from immune response as discussed in Dell et al.(1995). The terminal GalNAc residues may also target cancer cellstowards asialoglycoprotein receptors of liver (Yang et al., 2000). Bypreventing LacdiNAc-biosynthesis in cancer cells the metastaticpotential and possibly the malignancy of the cancer is reduced. Thepresent invention is especially directed to the prevention of lacdiNAcbiosynthesis for prevention of metastatic potential of cancer cells. Theoptimal antigenic epitopes are not designed as metastasis inhibitors.The vaccine type approach uses really low amounts of lacdiNAc structuresso that selectin mediated cell adhesion, which is necessary also fornormal immune system and leukocyte function, is not prevented.

The pharmaceutical composition according to the invention may alsocomprise other substances, such as an inert vehicle, or pharmaceuticallyacceptable carriers, preservatives etc., which are well known to personsskilled in the art.

The substance or pharmaceutical composition according to the inventionmay be administered in any suitable way. Methods for the administrationof therapeutic antibodies or vaccines are well-known in the art.

The term “treatment” used herein relates to both treatment in order tocure or alleviate a disease or a condition, and to treatment in order toprevent the development of a disease or a condition. The treatment maybe either performed in a acute or in a chronic way.

The term “patient”, as used herein, relates to any mammal in need oftreatment according to the invention.

When a cancer specific oligosaccharide or compound specificallyrecognizing cancer specific oligosaccharides of the invention is usedfor diagnosis or typing, it may be included, e.g., in a probe or a teststick, optionally in a test kit. When this probe or test stick isbrought into contact with a sample containing antibodies from a cancerpatient or cancer cells or tissue of a patient, components of a cancerpositive sample will bind the probe or test stick and can be thusremoved from the sample and further analyzed.

In the present invention the term “cancer” means “tumor” or “cancercells”. The term “tumor” means solid multicellular tumor tissues.Furthermore the term “tumor” means herein premalignant tissue, which isdeveloping to a solid tumor and has cancer specific characteristics. Theterm “tumor” is not referring herein to a single cell cancer such as aleukaemia or to cultured cancer cells or a cluster of such cells. Theexpression “cancer cells” means herein cells in tumor or single cancercells such as leukaemia cells or any other type of malignant cells,which are developing to a cancer or tumor form and have cancer or cancerspecific characteristics.

Enzymatic Synthesis of lacdiNAc Structures and Fucosylated lacdiNAcStructures and Analogs Thereof.

Terminal lacdiNac epitope can be synthesized by using large amounts ofβ4-galactosyltransferase (e.g. a commercially available bovine milkgalactosyltransferase) and molar excess of UDP-GalNAc in comparison tothe acceptor. For example incubations of UDP-GalNAc withGlcNAcβ3Galβ4Glc with relatively large amounts ofβ4-galactosyltransferase as described in (Nyame, et al 2000) givesGalNAcβ4GlcNAcβ3Galβ4Glc.

The reactions can also be performed by novel recombinant form ofgalactosyltransferase which can transfer GalNAc effectively fromUDP-GalNAc (Ramakrishnan et al., 2001).

The GalNAcβ4GlcNAc epitope or its sialylated derivativeNeuNAcα3GalNAcβ4GlcNAc can be fucosylated by several types ofα3-fucosyltransferases, for example by fucosyltrasferases from humanmilk essentially as described in Bergwerff et al. 1993 or byfucosyltransferase VI.

Production of Novel GalN Derivatives, Especially lacdiNac Structures andAnalogs Thereof

Present invention is also directed to novel pathways of enzymaticsynthesis. These can be used in vitro for production oflacdiNac-structures, related structures and analogs. Prior art hasdescribed glycosidase reactions on hexosamines and use of hexosaminedonors for glycosidase reactions. The present invention is directed tothe use of terminal hexosamines especially galactosamine andGalNβ4Glc(NAc)-terminal structures as acceptors for variousglycosyltransferases which normally use acceptor structures comprisingfor example terminal Gal or GalNAc residue.

These reactions have not previously been described and the positivecharge of amine group close to negatively charged nucleotide sugar donorcould have prevented the reactions. The charged aminogroup could alsoprevent recognition by enzymes requiring hydroxyl group in 2-position ofthe acceptor or cause undesired irreversible binding to the active siteof the enzyme. The present invention shows for the first time that thenovel glycosyltransferase reactions are possible and that the reactionsare possible even by mammalian glycosyltransferases. In comparison toglycosidase catalysed reactions the glycosyltransferase reactions arespecific forming in general only one type of products from a pair ofdonor and acceptor substrates, while the glycosidase catalysedtransglycosylation reaction yield in general several products.

Previously lacdiNAc derivatives have been synthesized by forcing theenzymes using Gal acceptors to use GalNAc. Yields in these methods arein general very poor. Some of the reactions with certain transferasesmay not be more effective than reactions with terminal GalNAc, but thepresence of amine allows specific synthesis of amine derivatives oranalogs of natural Gal/GalNAc sequences.

The novel reactions also reveal potential biosynthetic pathways to novelmalignant or disease associated antigenic structures, which have notbeen characterized from normal tissues. Especially novel blood grouprelated antigens are produced by natural glycosylation enzymes.

Preferred reactions include glycosyltransferase reactions to 3, 4, or 6position of the terminal hexosamine, preferably galactosamine and morepreferably to 3 or 6 position of the galactosamine and most preferablyto 3-position of galactosamine. Preferred reactions include:α3-sialyltransferase reactions, α6-sialyltransferase reactions,α3-galactosyltransferase reactions, α3-N-acetylgalactosaminyltransferasereactions, α3-galactosaminyltransfer reactions, β3-N-acetylglucosaminylreactions, β6-N-acetylglucosaminyl reactions, β3-N-acetylgalactosamiylreactions, β3-glucuronyltransferase reactions. Most preferred reactionsinclude α3-sialyltransferase reactions, α3-glalactosaminyltransferreactions and α3-galactosyltransferase reactions.

Preferred synthesis reactions are according to the formulaSAC-donor+GalNβ3/4→SACyxGalNβ3/4  (I)wherein y is α- or β-linkage and independently x is linkage position 3,4, or 6, SAC is sialic acid, or Hex(A)_(s1)[N(Ac)_(s2)]_(s3) wherein Hexis Gal or Glc and s1, s2, s3 are independently 0 or 1, with the provisothat when s1 is 1, then s3 is 0. When s1 is 1, the SAC-structurecomprises hexuronic acid structure, preferrably GlcA. When s3 is 1 ands2 is 1, the SAC-structure is GlcNAc or GalNAc, and when s2 is 0 thestructure is GalN or GlcN. GalNβ3/4 indicates nonreducing terminal GalNwhich is β3- or β4-linked to a hexose or hexosamine or hexosaminederivative, preferrably Gal, GalN, GalNAc, Glc, GlcN, or GlcNAc. In apreferred embodiment GalNβ4 is part of non reducing terminal structureGalNβ4GlcNAc or GalNβ4Glc, for example GalNβ4GlcNAcβ3Galβ4Glc.

The present invention is further directed to novel carbohydratesubstances according to the formulaGal[N(Am)_(s2)]_(s3)α3GalN(Am)_(s2)β3/4  (IV)wherein Am is a derivatization residue of amine amine group, with theprovision that Am is not acetyl (Ac) or an imidogroup, preferably Am iscarboxylic acid forming amide with the GalN residue such as formamide,propanoic acid amide, and other alkyl amides, and cyclic amidesincluding amides of cyclohexane radical comprising carboxylic acid andamides with aromatic hydrocarbons, for example amides of benzoic acids.Acetyl group is not preferred for the analog substances as it is presentin natural oligosaccharide sequences, bulky imidogroups such asphtalimido-group are not preferred because of the large size of thegroup which would change too much the conformation of the analog s2, s3are independently 0 or 1.

More preferred substances include terminal oligosaccharide sequencesGal[N(Am)_(s2)]_(s3)α3GalN(Am)_(s2)β4GlcNAC andGal[N(Am)_(s3)]α3GalN(Am)_(s2)β4Glc And most preferred terminaloligosaccharide sequences includes Galα3GalN(Am)_(s2)β4GlcNAc,Galα3GalN(Am)_(s2)β4Glc, and Galα3GalNβ4GlcNAc and Galα3GalNβ4Glc.

The novel substances are especially useful for use as antigens andimmunization. The rare and mostly non-natural or pathogenesis relatedstructures are useful immunogens which can be used for inducingproduction of antibodies which also recognize related structures. Aminecontaining substances are also useful starting materials for productionof further analogs, for testing as glycosidase substrates and/orinhibitors and testing as analogs of natural oligosaccharide sequencesfor binding of animal or plant lectins.

The present invention is also directed to combined reactions in whichUDP-GalN is first transferred to acceptor, for example to GlcNAc orGlucose or non-reducing end terminal GlcNAc or glucose and in the samereaction vessel the GalN is modified to 3, 4, or 6-position, preferablyto 3, or 6 position and most preferably to 3 position. In anothercombined reaction UDP-GalN is formed simultaneously in the same reactionwith two glycosyltransferases. Methods to produce UDP-GalN in situ forthe reaction have been previously described.

The present invention preferably uses a simplified process in whichUDP-GalN is generated from GalN1-phosphate and UDP-Glc. Preferredembodiment about synthesis of lacdiNAc-type structures includeN-acetylation of the hexosamine preferably to N-acetylhexosamine such asGalN to GalNAc.

In a preferred embodiment, analogs of an N-acetylhexosamine or hexosecomprising oligosaccharides are produced, preferrably lacdiNAc analogproducts are desired. The present invention is also directed toproduction of amine derivatives of the hexosamine, preferred amineanalogs or derivatives of hexosamine include amides such as formamide,propanoic acid amide, and other alkyl amides, and cyclic amidesincluding amides of cyclohexane radical comprising carboxylic acid andamides with aromatic hydrocarbons, for example amides of benzoic acids.

In a preferred embodiment a hexosamine is transferred to a hexosamine bya glycosyltransferase, preferably galactosamine is transferred togalactosamine, most preferably the product GalNα3GalNβ4GlcNAc is formed,this product can be further modified to GalNAcα3GalNAcβGlcNAc or analogsaccording to the present invention by derivatization of aminogroups.

In a separate embodiment UDP-GalN is transferred by aα-galactosyltransferase, or α-GalNActransferase, preferably byα3-galactosyltransferase, to Galβ-terminal containing acceptor. Theamine groups can be further derivatized to N-acetyl groups or analogscomprising derivatized aminen group. Preferrably products of the processare for example GalNAcα3Galβ4GlcNAc, GalNα3Galβ4GlcNAc,GalNAcα3(Fucα2)Galβ4, GalNα(Fucα2)3Galβ4, GalNAcα3(Fucα2)Galβ3,GalNα(Fucα2)3Galβ3, GalNAcα3(Fucα2)Galβ4GlcNAc,GalNα(Fucα2)3Galβ4GlcNAc, which corresponds to human A/B-blood groupsantigen terminal structures and analogs.

The present invention is further directed to novel carbohydratesubstances according to the formulaGalN(Am)_(r1)α3(Fucα2)_(r2)Galβ3/4  (V)wherein r1 and r2 are independently 0 or 1, Am is a derivatizationresidue of amine group, with the provision that Am is not acetyl (Ac) oran imidogroup, preferably Am is carboxylic acid forming amide with theGalN such as formamide, propanoic acid amide, and other alkyl amides,and cyclic amides including amides of cyclohexane radical comprisingcarboxylic acid and amides with aromatic hydrocarbons, for exampleamides of benzoic acids. Acetyl group is not preferred for the analogsubstances as it is present in natural oligosaccharide sequences, bulkyimidogroups such as phtalimido-group are not preferred because of thelarge size of the group which would change too much the conformation ofthe analog.

More preferred substances includes terminal oligosaccharide sequences

-   GalNα3Galβ4GlcNAc and GalNα3Galβ4Glc-   GalNα3(Fucα2)Galβ4GlcNAc and GalNα(Fucα2)3Galβ4Glc-   GalNAmα3(Fucα2)Galβ4GlcNAc and GalNAmα(Fucα2)3Galβ4Glc-   GalNAmα3Galβ4GlcNAc and GalNAmα3Galβ4Glc.

And most preferred substances includes terminal oligosaccharidesequences

-   GalNAmα3(Fucα2)Galβ4GlcNAc and GalNAmα(Fucα2)3Galβ4Glc-   GalNAmα3Galβ4GlcNAc and GalNAmα3Galβ4Glc.

The novel substances are especially useful for use as antigens andimmunization. The rare and mostly non-natural or pathogenesis relatedstructures are useful immunogens which can be used for inducingproduction of antibodies which also recognize related structures. Aminecontaining substances are also useful starting materials for productionof further analogs, for testing as glycosidase substrates and/orinhibitors and testing as analogs of natural oligosaccharide sequencesfor binding of animal or plant lectins.

Present invention is specifically directed to the use of novelsubstances according to the present invention for immunization, and forscreening of binding of lectins and antibodies. The present invention isespecially directed to screening of specificities of antibodies bindingto blood group antigens and screening related antibodies.

The present invention is specifically directed to the diagnostic and/ortreatment of oral cancers including preferably cancer of larynx andleukemia-type cancers which express oligosaccharide sequences accordingto the present invention. The present invention is also specificallydirected to the treatment according to the present invention for anytype of cancer which has surface expression of the lacdiNAc structuresaccording to the present invention. In another preferred embodiment thepresent invention is directed to the treatment of cancers from tissueswhich normally express and secrete proteins comprising lacdiNAcsequences, such tissues include glycoprotein hormone secreting tissues.

Glycolipid and carbohydrate nomenclature is according to recommendationsby the IUPAC-IUB Commission on Biochemical Nomenclature (Carbohydr. Res.1998, 322:167; Carbohydr. Res. 1997, 297:1; Eur. J. Biochem. 1998,257:29).

It is assumed that Gal, Glc, GlcNAc, and NeuNAc are of theD-configuration, Fuc of the L-configuration, and that all monosaccharideunits are in the pyranose form. Glucosamine is referred as GlcN andgalactosamine as GalN. Glycosidic linkages are shown partly in shorterand partly in longer nomenclature, the linkages α3 and α6 of theNeuNAc-residues mean the same as α2-3 and α2-6, respectively, and β1-3,β1-4, and β1-6 can be shortened as β3, β4, and β6, respectively.Lactosamine or N-acetyllactosamine or Galβ3/4GlcNAc means either typeone structure residue Galβ3GlcNAc or type two structure residueGalβ1-4GlcNAc, and sialic acid is N-acetylneuraminic acid or NeuNAc, Lacrefers to lactose and Cer is ceramide. SA refers to sialic acid such asNeuNAc. The cancer associated disaccharide sequence according to theinvention, GalNAcβ4GlcNAc, is referred as lacdiNAc or LacdiNAc.

The present invention is further illustrated in examples, which in noway are intended to limit the scope of the invention:

EXAMPLES Example I

Methods for Analysis of MMP-9

Isolation of MMP-9

MMP-9 was isolated from U-937 cells by sequential CM-cellulose (CM-52)chromatography followed by DEAE/Red Sepharose chromatography (Saarinenet al, 1999).

4-Vinylpyridine Alkylation of MMP-9

MMP-9 was concentrated and desalted by reversed-phase chromatography(RP-HPLC) on a 2.1×150-mm Poros® R2 column by elution with a lineargradient of acetonitrile (3-100% in 15 min) in 0.1% trifluoroaceticacid. Chromatography was performed at a flow rate of 1 ml/min andelution was monitored by UV absorbance at 214 nm. The eluted MMP-9 wasvacuum-dried and subjected to alkylation as follows: a sample wasdissolved in 80 μl of 6M guadine hydrochloride, 2 mM EDTA, 0.5M Tris pH7.5, reduced by addition of 5 μl of 0.6M DTT, and incubated for 20minutes at room temperature. After reduction, 1 μl of 4-vinylpyridinewas added, followed by alkylation for 15 minutes at room temperature.

The reaction was quenched by addition of 5 μl of 0.6M DTT. The alkylatedMMP-9 was immediately desalted by RP-HPLC, as described above.

Trypsin Digestion

2.5 μg (100 pmol) of alkylated MMP-9 was vacuum-dried and dissolved in40 μl of 50 mM ammonium bicarbonate buffer containing 1.66 ng/μl oftrypsin (Promega sequencing grade). Digestion was performed overnight at37 C.

Mass Spectrometry

MALDI:TOF MS was performed on a Biflex (Bruker Franzen Analytik) time offlight instrument equipped with a nitrogen laser operating at 337 m. Thetotal MMP-9 N-glycans, as well as the results of the NDV sialidasereactions were analysed in the linear positive ion delayed extractionmode using 2,4,6-trihydroxyacetophenone (Fluka Chemie AG, 3 mg/ml inacetonitrile/20 mM aqueous diammonium citrate, 1:1, v/v) as the matrix.The C perfringens sialidase and the fucosidase treated glycans wereanalysed in the reflector positive ion delayed extraction mode using2,5,-dihydrobenzoic acid (10 mg/ml) as the matrix. The spectra wereexternally calibrated with dextran 5000 (Fluka Chemie AG).

Electrospray ionization (ESI) mass spectra were collected using aMicromass Q-TOF hybrid quadrupole time-of-flight mass spectrometer(Micromass, UK). Ionization was accomplished by directing the LC eluentthrough a nanospray ion source equipped with a silica capillary needle20 μm i.d., 10 μm tip opening, gold-coated from the distal end (NewObjective Inc), operating at 2.2 kV.

Localization of Glycosylation Sites by Nanoflow LC/MS.

A sample of MMP-9 tryptic peptides (1 pmol) was separated by microborereversed-phase HPLC on a 0.075×150 mm PepMap column (NAN75-15-03-C18-PM,LC Packings) by elution with a linear gradient of acetonitrile (4-40% in30 min) in 0.1% formic acid. Chromatography was performed at a flow rateof 250 nl/min and the UV absorbance at 214 nm was recorded. LC wasdirectly coupled to a Micromass Q-TOF mass spectrometer. The massspectrometer was set to scan the HPLC eluent at both low and high conesettings to facilitate identification of the glycosylated components, asdescribed previously (Carr et al., 1993). A low cone scan was acquiredwith a cone voltage of 35 V, scanning over a mass range of m/z 100 to2500, thus providing a mass spectrum of the eluting components. A highcone scan was acquired with a cone voltage of 120 V, which applies ahigh collisional excitation potential to all ions during their entryinto the mass spectrometer. This leads to collision-inducedfragmentation of the entering ions prior to mass separation. During ahigh cone potential scan the mass spectrometer was set to scan from m/z100 to 1000. Reconstituted chromatograms were then created for 204.1(oxonium ion of HexNAc) and m/z 292.15 (oxonium ion of Neu5Ac) and m/z366.1 (oxonium ion of Hex-HexNac), thus providing chromatogramsselective for eluting glycopeptides.

Analysis of LacdiNAc Structures from Leukaemia Cells

To demonstrate abnormal expression of LacdiNAc sequence on cancer cellsmetalloproteinase-9 (MMP-9) was isolated from leukaemia cells (U-937),and N-glycosidic glycans were liberated with N-glycosidase F. The glycanfraction was analyzed with MALDI-TOF MS in trihyxroacetophenone matrix(FIG. 1A), which has been shown to cause negligible fragmentation ofsialic acid residues. The assignment of the monosaccharide compositionsas well as the proposed structures according to the subsequentglycosidase treatments are shown in Table 1. The relative abundances ofthe components are indicated as well, as oligosaccharide analysis inMALDI-TOF has been shown to be relatively quantitative. The mostabundant glycan species was assigned to [M+Na]⁺ of(Hex)₅(HexNAc)₄(Fuc)₃. Knowing the typical N-glycan structures, wetentatively assigned this structure as a trifucosylated diantennarycomplex-type glycan. Other major species were identified as sialylated,difucosylated diantennary complex-type glycan, and trifucosylateddiantennary complex-type glycan carrying GalNAc instead of Gal as aterminal monosaccharide (so called LacdiNAc structure) in one antennae.These assignments were found to be correct, as shown by sequentialglycosidase treatments. For comparison the matrix metalloproteinaseMMP-9 structures in non-malignant white blood cells (leukocytes) hasbeen determined, the protein did not contain lacdiNAc sequences (Rudd etal, 1999).

Structural characterization of the glycans was performed by sequentialglycosidase treatments, which were monitored by MALDI-TOF MS. Todistinguish the two plausible Neu5Ac linkages (α2-3 vs. α2-6), theglycan mixture was treated with NDV neuraminidase, an enzyme strictlyspecific for α2-3neuraminic acid. Partial cleavage could be observed(FIG. 1B) that may arise from SA α2-3GalNAc bond, which has been shownto resist the action of NDV sialidase. Other possibility is the presenceof both α2-3 and α2-6 linked SA. Treatment of the N-glycan fraction witha broad specificity neuraminidase (Clostridium perfingens) resulted infollowing changes (FIG. 1C): Signals at m/z 2247.3, 2287.9, and 2392.3disappeared, and signals appeared at m/z 1809.70, 1850.78, 1891.75,1955.77, and 1996.79. The results indicate the presence ofdifferentially fucosylated forms of normal biantennary N-glycans,biantennary N-glycans carrying LacdiNAc structure (GalNAcβ1-4GlcNAc) inone of the antennae, and a minor amount of N-glycans carrying LacdiNAcstructure in both of the antennae. Treatment of the sample by Almondmeal α1-3(4)fucosidase (which removes the α1-3(4) linked fucose residuesfrom the antennae, but does not cleave the α1-6 linked fucose residuefrom the N-glycan core) led to appearance of signals at 1809.80,1850.78, and 1891.84 (67%, 27% and 6% of intensity). These resultsindicate that approximately 30% of the glycans carry LacdiNAc sequencein either one or both of the antennae. This result is well in accordancewith the LC-ESI MS data presented below.

MMP-9 was isolated from U-937 cells, alkylated, and digested withtrypsin. To identify the glycopeptides, we conducted an LC/MS analysisof tryptic peptides from MMP-9, using stepped cone voltages to obtainboth the total ion chromatogram (FIG. 2A) and chromatograms showingpotential glycopeptides (FIGS. 2B, C and D). It should be noted that inthe chromatography setup used in this study (i.e. PepMap media run in0.1% formic acid-acetonitrile), the separation of glycopeptides isaffected by both the peptide moiety and the glycan moiety. In the morecommonly used trifluoroacetic acid-acetonitrile system, the separationof the glycopeptides is dominated by the peptide moiety alone. Thisleads to elution of the glycopeptides as two major peaks eluting at 23.1and 24.3 minutes.

The mass spectrum of the material eluting at 23.1 min is presented inFIG. 3A, and shows signals which could all be assigned to glycosylatedpeptide Trp¹¹⁶-Arg¹³⁴, carrying complex-type glycans (see Table 1). Themost intense ion in this spectrum at m/z 1143.49 is assigned to [M+4H]⁴⁺of Trp¹¹⁶-Arg¹³⁴, carrying (Hex)₅(HexNAc)₄(Fuc)₃. The mass spectrum ofthe material eluting at 24.3 min is presented in FIG. 3B. The mostintense ion in this spectrum at m/z 1179.54 is assigned to [M+4H]⁴⁺ ofTrp¹¹⁶-Arg¹³⁴, carrying (Hex)₅(HexNAc)₄(Fuc)₂(SA)₁.

The present invention shows that U-937-cell derived MMP-9 carriesLacdiNAc structures in large fraction (approximately 30%) of itsN-glycans. The presence of LacdiNAc structures was verified by twoindependent methods, namely MALDI-TOF analysis of liberated N-glycans aswell as LC-ESI MS of intact glycopeptides. The assignments of thestructures were further confirmed by sequential glycosidase treatments.The methods used in this study have been verified by several approachesusing both known natural structures as well as syntheticoligosaccharides.

Sialylated N-glycans that contain LacdiNAc sequences from larynx cancer.Human melanoma cell (RPMI-7932 and RPMI-7951) membrane protein N-glycansthat contain LacdiNAc, sialyl-LacdiNAc, and fucosyl-LacdiNAc sequences.

Example II

Analysis of Solid Tumor and Melanoma Cell Membranes

Cancer sample material. The larynx cancer sample was a formalin-fixedtumor specimen collected during a surgical operation. Prior to glycanisolation, proteins were enriched by chloroform-methanol extractionessentially as described in (Manzi et al., 2000). Quantitativeextraction of glycoproteins was confirmed by radioactively labelledglycoprotein standards (not shown).

Isolation of glycans from chloroform-methanol extracted proteins.Glycans were detached from sample glycoproteins by non-reductiveβ-elimination and purified by chromatographic methods.

Isolation of human melanoma cell membrane proteins. Human melanoma cells(RPMI-7932 and RPMI-7951) were washed with phosphate buffered saline(PBS) at room temperature, scraped off from cell culture dishes, andcollected by centrifugation. Thereafter, the purification processcontinued at +0-+4° C. The cells were incubated in hypotonic buffer, 25mM Tris-HCl pH 8.5, broken by homogenisation, and brought back toisotonic buffer by the addition of NaCl to 150 mM. The nuclei wereseparated from the bulk of cell membranes by low-speed centrifugation,which was monitored by microscopy. The supernatant, containing themembranes and cytosolic material, was centrifuged at 40,000 g. Thepellet (the membrane preparation) was homogenized in detergent buffercontaining 25 mM Tris-HCl pH 8.5, 150 mM NaCl, and 1% (w/v) TritonX-100. After incubation, the preparate was centrifuged at 100,000 g andthe supernatant, containing the detergent extracted membrane proteins,was collected. Buffer salts and the detergent were removed by coldacetone precipitation as in (Verostek et al., 2000).

Isolation of membrane protein N-glycans. N-glycans were detached frommembrane glycoproteins with Chryseobacterium meningosepticumN-glycosidase F (Calbiochem, USA) essentially as in (Nyman et al., 1998)and purified essentially as in (Verostek et al., 2000; Packer et al.,1998). N-glycans from RPMI-7951 cells were additionally passed throughcolumns of 1) AG-50W strong cation exchange material and 2) C₁₈ silicain water, whereas N-glycans from RPMI-7932 cells were not. The N-glycanswere separated into sialylated and non-sialylated fractions withgraphitised carbon columns essentially as in (Packer et al., 1998).

MALDI-TOF MS. MALDI-TOF mass spectrometry was performed with aVoyager-DE STR BioSpectrometry Workstation, essentially as in (Saarinenet al., 1999; Papac et al., 1996; Harvey, 1993).

Exoglycosidase digestions. All exoglycosidase reactions were performedessentially as described in (Nyman et al., 1998; Saarinen et al., 1999)and analysed by MALDI-TOF MS. The enzymes and their specific controlreactions with characterised oligosaccharides were: Arthrobacterureafaciens sialidase (recombinant, E. coli; Glyko, UK) digested bothNeu5Acα2-3Galβ1-4GlcNAc-R and Neu5Acα2-6Galβ1-4GlcNAc-R inoligosaccharides; β-N-acetylglucosaminidase (Streptococcus pneumoniae,recombinant, E. coli; Calbiochem, USA) digested GlcNAcβ1-6Gal-R but notGalNAcβ1-4GlcNAcβ1-3/6Gal-R; β-N-acetylhexosaninidase (Jack bean;Calbiochem, USA) digested both GlcNAcβ1-6Gal-R andGalNAcβ1-4GlcNAcβ1-3/6Gal-R; α1,3/4-fucosidase (Xanthomonas sp.;Calbiochem, USA) digested Galβ1-4(Fucα1-3)GlcNAc-R but notFucα1-2Galβ1-3GlcNAc-R Control digestions were performed in parallel andanalysed similarly to the analytical exoglycosidase reactions.

Results

LacdiNAc containing sialylated N-glycans from larynx cancer samples. Thesialylated glycans from larynx cancer samples were effectivelydesialylated with Arthrobacter ureafaciens sialidase. Desialylation wasmonitored by MALDI-TOF MS (not shown). The desialylated glycans werefirst digested with β-N-acetylglucosaminidase at enzyme concentrationsthat specifically hydrolyse terminal β-GlcNAc residues but not β-GalNAcresidues. The further addition of β-N-acetylhexosaminidase removed 2HexNAc residues from one of the N-glycans, indicating the presence of aGalNAcβ1-4GlcNAc sequence, which is resistant to the action ofβ-N-acetylglucosaminidase but is completely digested withβ-N-acetylhexosaminidase. Upon β-N-acetylhexosaminidase digestion, therelative signal intensity of a peak at m/z 1850.38/1850.16,corresponding to the ion [Hex₄HexNAc₅Fuc₁+Na]⁺ (calc. m/z 1850.67), wassignificantly decreased. Simultaneously, the relative signal intensityof a peak at m/z 1444.30/1444.15, corresponding to the ion[Hex₄HexNAc₃Fuc₁+Na]⁺ (calc. m/z 1444.51), was increased, while therewas no increase in the relative signal intensity of the incompletelydigested form at m/z 1647.34/1647.19, corresponding to the ion[Hex₄HexNAc₄Fuc₁+Na]⁺ (calc. m/z 1647.59). The one observed peakcorresponding to a sialylated form of the LacdiNAc containing N-glycanin question, namely the ion at m/z 2118.09

([NeuAc₁Hex₄HexNAc₅Fuc₁-H]⁻; calc. m/z 2118.93), contains only onesialic acid residue. However, the present data cannot exclude thepresence of differently sialylated forms in the original sample.Importantly, no evidence of LacdiNAc containing glycans could beobtained in similarly analysed samples from many healthy tissues.

RPMI-7932 and RPMI-7951 human melanoma cell membrane proteindesialylated N-glycans. The sialylated N-glycans, comprising tomembrane-associated sialylated N-glycans, were effectively desialylatedwith Arthrobacter ureafaciens sialidase. Desialylation was monitored byMALDI-TOF MS (data not shown). The desialylated N-glycans were firstdigested with β-N-acetylglucosaminidase at enzyme concentrations thatspecifically hydrolyse terminal β-GlcNAc residues but not β-GalNAcresidues. The further addition of β-N-acetylhexosaminidase removedHexNAc residues from some of the N-glycans. Below are the examples ofthe β-N-acetylhexosaminidase sensitive and β-N-acetylglucosaminidaseinsensitive structures, from which the enzyme removed exclusively either2 or 4 HexNAc residues at a time. Taken together, this indicates thatthese structures contain GalNAcβ1-4GlcNAc sequences, which are resistantto the action of β-N-acetylglucosaminidase but are completely digestedwith β-N-acetylhexosamidase.

LacdiNAc Containing N-Glycans from RPMI-7932 Human Melanoma CellMembrane Proteins (FIG. 5.-7.):

A. Upon β-N-acetylhexosaminidase digestion, a peak at m/z 1704.71,corresponding to the ion [Hex₄HexNAc₅+Na]⁺ (calc. m/z 1704.61), wastransformed into a peak at m/z 1298.53, corresponding to the ion[Hex₄HexNAc₃+Na]⁺ (calc. m/z 1298.45), while there was no increase inthe relative signal intensity of the incompletely digested form at m/z1501.62, corresponding to the ion [Hex₄HexNAc₄+Na]⁺ (calc. m/z 1501.53).

B. Upon β-N-acetylhexosaminidase digestion, the relative signalintensity of a peak at m/z 1850.73/1850.72, corresponding to the ion[Hex₄HexNAc₅Fuc₁+Na]⁺ (calc. m/z 1850.67), was significantly decreased.Simultaneously, the relative signal intensity of a peak at m/z1444.59/1444.58, corresponding to the ion [Hex₄HexNAc₃Fuc₁+Na]⁺ (calc.m/z 1444.51), was significantly increased, while there was no increasein the relative signal intensity of the incompletely digested form atm/z 1647.66, corresponding to the ion [Hex₄HexNAc₄Fuc₁+Na]⁺ (calc. m/z1647.59).

C. Upon β-N-acetylhexosaminidase digestion, a peak at m/z 1891.78,corresponding to the ion [Hex₃HexNAc₆Fuc₁+Na]⁺ (calc. m/z 1891.69), wascompletely transformed into peaks at m/z 1079.47, corresponding to theion [Hex₃HexNAc₂Fuc₁+Na]⁺ (calc. m/z 1079.38), and m/z 1485.65/1485.61,corresponding to the ion [Hex₃HexNAc₄Fuc₁+Na]⁺ (calc. m/z 1485.53),while no evidence could be found of the possible incompletely digestedforms at calc. m/z 1688.61 [Hex₃HexNAc₅Fuc₁+Na]⁺ or calc. m/z 1282.45[Hex₃HexNAc₃Fuc₁+Na]⁺. Upon α1,3/4-fucosidase digestion, the peak at m/z1485.61 was partly transformed into a peak at 1339.52, corresponding tothe ion [Hex₃HexNAc₄+Na]⁺ (calc. m/z 1339.48).

D. Upon β-N-acetylhexosaminidase digestion, the relative signalintensity of a peak at m/z 2215.85/2215.84, corresponding to the ion[Hex₅HexNAc₆Fuc₁+Na]⁺ (calc. m/z 2215.80), was significantly decreased.Simultaneously, the relative signal intensity of a peak at m/z1809.71/1809.67, corresponding to the ion [Hex₅HexNAc₄Fuc₁+Na]⁺ (calc.m/z 1809.64), was significantly increased, while there was no increasein the relative signal intensity of the possible incompletely digestedform at m/z 2012.76, corresponding to the ion [Hex₅HexNAc₅Fuc₁+Na]⁺(calc. m/z 2012.72).

LacdiNAc Containing N-Glycan from RPMI-7932 Human Melanoma Cell LineMembrane Proteins (FIG. 8.-9.):

E. Upon β-hexosaminidase digestion, a peak at m/z 1501.52, correspondingto the ion [Hex₄HexNAc₄+Na]⁺ (calc. m/z 1501.53), was transformed into apeak at m/z 1095.36, corresponding to the ion [Hex₄HexNAc₂+Na]⁺ (calc.m/z 1095.37), while there was no significant increase in the relativesignal intensity of the incompletely digested form at m/z1298.48/1298.46, corresponding to the ion [Hex₄HexNAc₃+Na]⁺ (calc. m/z1298.45).

Conclusions

The present results indicate that human cancerous tissue containsglycoproteins that carry LacdiNAc sequences. More specifically, theresults show that a LacdiNAc containing a sialylated N-glycan,NeuAc₁Hex₄HexNAc₅Fuc₁, is expressed on glycoproteins of a larynx cancertumor specimen. Furthermore, the present results suggest the presence ofseveral LacdiNAc, sialyl-LacdiNAc, and/or fucosyl-LacdiNAc containingstructures among the sialylated N-glycans of RPMI-7932 melanoma cellmembrane proteins, and the presence of a sialyl-LacdiNAc containingN-glycan among the sialylated N-glycans of RPMI-7951 melanoma cellmembrane proteins. In structure C. of RPMI-7932 melanoma cell linemembrane protein desialylated N-glycans, namely Hex₃HexNAc₆Fuc₁, atleast one of the LacdiNAc units is originally sialylated, as theN-glycans were obtained through Arthrobacter ureafaciens sialidasedigestion from isolated sialylated N-glycans. Similarly, in structure C.of RPMI-7932 melanoma cell line membrane protein sialylated N-glycans,at least one of the two LacdiNAc sequences must be β-linked to one ofthe α-mannoses of the N-glycan core. Furthermore, α1,3/4-fucosidasedigestion revealed the presence of α1,3-fucosylated LacdiNAc sequencesin structure C. of RPMI-7932 melanoma cell line membrane proteinsialylated N-glycans. In the other structures, the presence ofHex-HexNAc sequences prevents these structure assignments. Themonosaccharide composition of structure E. of RPMI-7951 melanoma cellline membrane protein desialylated N-glycans, Hex₄HexNAc₄, suggests thepresence of a hybrid N-glycan that has a sialyl-LacdiNAc sequence on aMan₄GlcNAc₂ N-glycan core.

Example III

Anti-LacdiNAc Antibodies that Recognize GalNAcβ1-4GlcNAc Sequences onCancer Cells and Glycoconjugates, and Immunogenic OligosaccharideConjugates Used to Produce and Characterize Them

Antibodies. Anti-LacdiNAc antibodies are produced by immunogenicLacdiNAc conjugates by standard methods in experimental animalimmunology or screening antibody libraries by phage display or othermethods known in the art. The used possibly monoclonal antibodies areshown to be specific towards LacdiNAc by ELISA with specificoligosaccharide or oligosaccharide conjugate coated ELISA plates, or anyother suitable method that utilizes recognition of specificoligosaccharides or their conjugates. Antibodies that bind to theLacdiNAc antigen without binding to the respective LacNAc analog, aresuitable for theit intended use. The specific pairs of a LacdiNAcantigen and a LacNAc control antigen, suitable for conjugation asneoglycoproteins or other immunogenic conjugates, are for example asfollows: GalNAcβ14GlcNAcβ1-3Galβ1-R and Galβ1-4GlcNAcβ1-3Galβ1-R,GalNAcβ4GlcNAcβ1-6Galβ1-R and Galβ1-4GlcNAcβ1-6Galβ1-R,GalNAcβ1-4GlcNAcβ1-3GalNAcα1-R and Galβ1-4GlcNAcβ1-3GalNAcα1-R,GalNAcβ1-4GlcNAcβ1-6GalNAcα1-R and Galβ1-4GlcNAcβ1-6GalNAcα1R,GalNAcβ1-4GlcNAcβ1-2Manα1-R and Galβ1-4GlcNAcβ1-2Manα1-R,GalNAcβ1-4GlcNAcβ1-6Manα1-R and Galβ1-4GlcNAcβ1-6Manα1-R,GalNAcβ1-4GlcNAcβ1-4Manα1-R and Galβ1-4GlcNAcβ1-4Manα1-R, andcorresponding sialylated and/or fucosylated analogs of theabovementioned epitopes according to the invention, where R can be forexample lactose, any O- or N-glycan or glycolipid core structure, or aspacer that is used for the conjugation of the oligosaccharide to theneoglycoprotein or any other immunogenic carrier by methods known in theart.

In situ generation of LacdiNAc sequences on tissue sections. Tissuecontrols can be subjected to in situ LacdiNAc synthesis to serve aspositive controls in immunohistochemistry or other diagnosticapplications. The reaction can be facilitated for example by a mutantβ1,4-galactosyltransferase similar to the bovine milk enzyme Y289Lmutant described in (Ramakrishnan and Qasba, 2002) and by using reactionconditions essentially similar to ones described in the cited reference.Before the GalNAc transfer reaction, the tissue sections can beincubated with Jack bean β-galactosidase (Glyko, UK) at a concentrationof 0.5 U/ml, in 50 mM sodium acetate pH 4.0, at +37° C. overnight, afterwhich the glycosidase reaction solution is washed away from thesections. This will create additional acceptor sites for theGalNAc-transferase.

Use of antibodies in immunodiagnostic applications. The LacdiNAcspecific antibodies can be used in immunohistochemistry,immunodiagnostics, and other diagnostic applications according to thestandard immunological methods.

Example IV

In Vitro Lysis Assay to Detect Cytolytic Activity of the AntibodiesRecognizing lacdiNAc Structures According to the Present Invention.

Model cancer cells comprising lacdiNAc structures on cell surface areharvested to 80% confluent density and washed four times with HBSS(Hank's balanced salt solution). The viability of the cells isdetermined by trypen blue staining. Approximately 200 000 cells areincubated with antibodies binding the lacdiNAc-structures according tothe present invention on the cell surfaces. Rabbit complement is addedto one set of cells to a final dilution of 1:5 and the other set ofcells is adjusted to same volume with incubation media. The cells arefurther incubated for 1 h at 37 degree of Celsius. Finally, propiumiodide is added to final concentration of 20 micrograms per milliliterand the cells are analyzed for dye uptake. The cells are shown to belysed by the lacdiNAc binding antibodies but not by non-cancer bindingcontrol antibodies. The lacdiNac structures are available for antibodyrecognition and cytolysis on cell surface.

Example V

Example of α3-Sialyltransferase Reaction:

Molar excess of CMP-NeuNAc (20 micromol) and α3-sialyltransferase (1 U,ST3Gal III, rat liver, Calbiochem) is incubated overnight at 37 degreeof Celsius with GalNβ4GlcNAcβ3Lac (5 micromol) in 2.1 ml 50 mM MOPS-NaOHpH 7.4 containing 2 mg/ml BSA. Product NeuNAcα3GalNβ4GlcNAcβ3Galβ4Glc isformed quantitatively and purified by gel filtrationHPLC-chromatography. Mass spectrometry and NMR-analysis confirmed theexpected structure. MALDI-TOF mass spectrometry in negative linear moderevealed the product peak at m/z 998.36. The products can be optionallyN-alkylated or derivatized to an analog of α3-sialylated LacdiNAc.α3-sialylated lacdiNAc is obtained by N-acetylation of GalN to GalNAc by8 microliters of acetic anhydride in 200 microliters of 1 M NH₄HCO₃.

Examples of α3-Galactosyltransferase Reactions

Synthesis of Galα3GalNβ4GlcNAcβ3Lac from GalNβ4GlcNAcβ3Lac UDP-Gal (3micromol), GalNβ4GlcNAcβ3Lac (1.5 micromol), andα3-galactosyltransferase (0.1 U, Calbiochem) is incubated at 37 degreesof Celsius in 500 microliters 100 mM MES buffer pH 7.0 containing 20 mMMgCl₂. Product Galα3GalNβ4GlcNAcβ3Galβ4Glc was purified by gelfiltration HPLC-chromatography. The reaction was incubated at 37 degreesof Celsius for 4 days. MALDI-TOF mass spectrometry in positive ion moderevealed the expected product peak at m/z 891.2102. The structure of theproduct is confirmed by NMR-spectrometry.

Example VI

Synthesis of GalNα3GalNβ4GlcNAcβ3Lac from GlcNAcβ3Lac by TwoGalactosyltransferases and In Situ Donor Synthesis.

The reaction mixture containing 5 micromol of GlcNAcβ3Lac, 10 mM GalN-1P(Sigma), 20 mM UDP-Glc, 2.5 U galactos-1-phosphate-uridyltransferase,0.5 U β1-4-galactosyltransferase and 0.5 U α1-3-galactosyltransferase(Calbiochem, Calif., USA) is incubated in 1.0 ml 0.1 M HEPES pH 8.0containing 5 mM MgCl₂ and 5 mM β-mercaptoethanol. The reaction wasincubated at 37 degrees of Celsius for 4 days. MALDI-TOF analysis of thereaction products in positive ion mode revealed major product peak atm/z 890.2349. The structure of the product is confirmed byNMR-spectrometry.

TABLE 1 corresponding signal MMP-9 glycans, in MALDI peak 23.10 m mabundance % LacDiNac analysis assignment m/z z (observed) (calculated)(of total)* % (FIG. 1A)** Hex5HexNac4Fuc1 1070.201 4 4276.804 4276.81210.1 Hex4HexNac5Fuc1 1080.498 4 4317.992 4317.838  4.8  4.8Hex5HexNac4Fuc2 1106.763 4 4423.052 4422.87  13.4 1956.01Hex4HexNac5Fuc2 1117.036 4 4464.144 4463.896  7.8  7.8 1996.64Hex5HexNac4Fuc3 1143.247 4 4568.988 4568.928 21.2 2101.96Hex4HexNac5Fuc3 1153.518 4 4610.072 4609.954  8.1  8.1 2143.07 65.5 20.7MMP-9 glycans, peak 24.30 abundance % LacDiNac assignment m/z z m (oftotal)* % Hex5HexNac4SA1 1106.697 4 4422.788 4421.849  2.9Hex5HexNac4Fuc1SA1 1143.008 4 4568.032 4567.907 10.6 Hex4HexNac5Fuc1SA11153.318 4 4609.272 4608.964  3.1  3.1 Hex5HexNac4Fuc2SA1 1179.540 44714.16  4713.965 13.7 2247.27 Hex4HexNac5Fuc2SA1 1189.780 4 4785.12 4754.992  4.1  4.1 2288.22 34.5  7.2 total abundance (%) 100.0  27.9*Quantification based on LC/MS analysis **Analysed as [M + Na]^(+Ion)

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1. A method for diagnosing larynx cancer in a larynx tissue sample froma patient suspected of having larynx cancer comprising determining thepresence in said larynx tissue sample of an oligosaccharide sequenceaccording to Formula(Sac1)_(x)GalNAcβ4(Fucα3)_(y)GlcNAc  (I) wherein x and y are eachindependently 0 or 1 and Sac1 is NeuNAcα3 or NeuNAcα6, and wherein thedetermination comprises (a) contacting said larynx tissue sample with anantibody to said oligosaccharide sequence, and determining the presenceof a combination of said antibody bound to said oligosaccharidesequence, the presence of said oligosaccharide sequence being anindication of the presence of larynx cancer in said larynx tissuesample, or (b) releasing the oligosaccharide structures from said larynxtissue sample by enzymatic or chemical methods to form a fractioncontaining free oligosaccharide structures or conjugates from saidlarynx tissue sample, and determining the presence of saidoligosaccharide sequence in said fraction, the presence of saidoligosaccharide sequence in said fraction being an indication of thepresence of larynx cancer in said larynx tissue sample.
 2. The methodaccording to claim 1, wherein said antibody is specific tooligosaccharide sequence GalNAcβ4GlcNAc.
 3. The method according toclaim 1, wherein said antibody is specific to the oligosaccharidesequence according to Formula I.
 4. A method for diagnosing larynxcancer in a larynx tissue sample comprising determining the presence insaid larynx tissue sample of an oligosaccharide sequence according toFormula(Sac1)_(x)GalNAcβ4(Fucα3)_(y)GlcNAc  (I) wherein x and y are eachindependently 0 or 1 and Sac1 is NeuNAcα3 or NeuNAcα6, and wherein thedetermination comprises (a) contacting said larynx tissue sample with anantibody to said oligosaccharide sequence, and determining the presenceof a combination of said antibody bound to said larynx tissue sample,and (b) contacting a control larynx tissue sample of correspondinghealthy tissue with said antibody, and determining the presence of acombination of said antibody bound to said control larynx tissue sample,wherein a higher presence of said oligosaccharide sequence in saidlarynx tissue sample compared to said control larynx tissue sample isindicative of larynx cancer in said larynx tissue sample; or (c)releasing the oligosaccharide structures from said larynx tissue sampleby enzymatic or chemical methods to form a fraction containing freeoligosaccharide structures or conjugates from said larynx tissue sample,and determining the presence of said oligosaccharide sequence in saidfraction; and (d) releasing the oligosaccharide structures from saidcontrol larynx tissue sample by enzymatic or chemical methods to form afraction containing free oligosaccharide structures or conjugates fromsaid control larynx tissue sample, and determining the presence of saidoligosaccharide sequence in said fraction, wherein a higher presence ofsaid oligosaccharide sequence in the fraction obtained from said larynxtissue sample compared to the fraction obtained from said control larynxtissue sample is indicative of the presence of larynx cancer in saidsample.
 5. The method according to claim 4, wherein said antibody isspecific to the oligosaccharide sequence according to Formula I.
 6. Themethod according to claim 4, wherein said antibody is specific tooligosaccharide sequence GalNAcβ4GlcNAc.
 7. The method according toclaim 1, wherein the detected oligosaccharide sequence is on cell ortissue surface.
 8. The method according to claim 4, wherein the detectedoligosaccharide sequence is on cell or tissue surface.
 9. The methodaccording to claim 1, wherein in Formula (I), y is 1 only when x is 1.10. The method according to claim 4, wherein the presence of saidoligosaccharide sequence is determined in step c) or d) byNMR-spectroscopy, mass spectrometry or glycosidase degradation methods.11. The method according to claim 4, wherein the larynx tissue sample istaken from a patient suspected to suffer from larynx cancer.
 12. Themethod according to claim 4, wherein the larynx tissue sample is a tumorsample or a sample of larynx tissue that is suspected to be cancerous.13. A method for screening tumor or cancer tissue samples for thepresence of an oligosaccharide according to Formula(Sac1)_(x)GalNAcβ4(Fucα3)_(y)GlcNAc  (I) wherein x and y are eachindependently 0 or 1 and Sac1 is NeuNAcα3 or NeuNAcα6, and wherein thedetermination comprises (a) contacting said tissue sample with anantibody to said oligosaccharide sequence, and determining the presenceof a combination of said antibody bound to said tissue sample, and (b)contacting a control sample of corresponding healthy tissue with saidantibody, determining the presence of a combination of said antibodybound to said control sample, and comparing the level of saidoligosaccharide sequence in said tissue sample to that in said controlsample; or (c) releasing the oligosaccharide structures from said tissuesample by enzymatic or chemical methods to form a fraction containingfree oligosaccharide structures or conjugates from said tissue sample,and determining the presence of said oligosaccharide sequence in saidfraction; and (d) releasing the oligosaccharide structures from saidcontrol sample by enzymatic or chemical methods to form a fractioncontaining free oligosaccharide structures or conjugates from saidcontrol sample, determining the presence of said oligosaccharidesequence in said fraction, and comparing the level of saidoligosaccharide sequence in the fraction obtained from said tissuesample to that in the fraction obtained from said control sample. 14.The method according to claim 13, wherein said tumor or cancer tissue isfrom leukemia, melanoma or larynx cancer.
 15. A method for analysing aputative cancer sample comprising determining the presence in saidsample of an oligosaccharide sequence according to Formula(Sac1)_(x)GalNAcβ4(Fucα3)_(y)GlcNAc  (I) wherein x and y are eachindependently 0 or 1 and Sac1 is NeuNAcα3 or NeuNAcα6, wherein thedetermination comprises (a) contacting said putative cancer sample withan antibody binding to said oligosaccharide sequence, and determiningthe presence of a combination of said antibody and said sample, thepresence of said combination being an indication of the presence of saidoligosaccharide in said sample, or (b) releasing the oligosaccharidestructures of said putative cancer sample by enzymatic or chemicalmethods to form a fraction containing free oligosaccharide structures orconjugates from said sample, and determining the presence of saidoligosaccharide sequence in said fraction.
 16. The method according toclaim 15, wherein said putative cancer sample is from leukemia, melanomaor larynx cancer.
 17. The method according to claim 15, wherein theputative cancer sample is from a human or animal patient suspected tohave cancer.
 18. The method according to claim 15, wherein the methodcomprises a further step of purification, detection or quantitation ofan antibody binding to the oligosaccharide sequence according to FormulaI from the serum of a patient, when the presence of an oligosaccharidesequence according to Formula I is detected in said sample.
 19. Themethod according to the claim 13, wherein the method comprises a furtherstep of purification, detection or quantitation of an antibody bindingto the oligosaccharide sequence according to Formula I from the serum ofa patient, when the presence of an oligosaccharide sequence according toFormula I is detected in said sample.
 20. The method according to theclaim 15, wherein step (a) is performed as an in vitro cytolysis assayof cancer cells with antibodies binding to the oligosaccharide sequenceaccording to Formula I.